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-Lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human -lactalbumin made lethal to tumor cells)

¹ Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, and
² Department of Biophysical Chemistry, Lund University, Lund, Sweden
³ Departments of Veterinary Biosciences and Chemistry, The Ohio State University, Columbus, Ohio 43210, USA
⁴ Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208-2436, USA

Reprint requests to: Catharina Svanborg, Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden; e-mail: Catharina.Svanborg{at}mig.lu.se; fax: 46-46-137468.

HAMLET (human -lactalbumin made lethal to tumor cells) is a complex of human -lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from -lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of -lactalbumin is sufficient to induce cell death. We used the bovine -lactalbumin Ca²⁺ site mutant D87A, which is unable to bind Ca²⁺, and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine -lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca²⁺site, as HAMLET maintained a high affinity for Ca²⁺ but D87A-BAMLET was active with no Ca²⁺ bound. We conclude that partial unfolding of -lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca²⁺-binding site is not required for conversion of -lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca²⁺site.

Keywords: -Lactalbumin; Ca²⁺-binding site; protein folding; HAMLET; tumor cell death

Abbreviations: CD, circular dichroism • ANS, 8-Anilinonaphtalene-1-sulfonic acid • Tris, tris(hydroxymethyl)aminomethane • EDTA, ethylenediamine tetra acetic acid • FPLC, fast protein liquid chromatography • PBS, phosphate-buffered saline • UV, ultraviolet • DEAE, diethylaminoethyl • quin 2, 2–[[2–[bis(carboxymethyl)amino-5-methylphenoxy]methyl]-6-methoxy-8–[bis(carboxymethyl)amino]quinoline

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				L. Gustafsson, O. Hallgren, A.-K. Mossberg, J. Pettersson, W. Fischer, A. Aronsson, and C. Svanborg HAMLET Kills Tumor Cells by Apoptosis: Structure, Cellular Mechanisms, and Therapy J. Nutr., May 1, 2005; 135(5): 1299 - 1303. [Abstract] [Full Text] [PDF]

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